Sang Joon Won/sandbox 1

=Lactose Permease=



Background
The lactose permease (LacY) is arguably a paradigm for secondary transporter proteins. This membrane protein has been studied for decades to understand the detailed mechanism of energy transduction and translocation reactions. This protein serves as the lactose and hydrogen ion (H+) symporter, utilizing the free energy released from downhill movement of H+ to concentrate lactose inside a cell. Lactose is a type of sugar called disaccharide and LacY is generally specific for disaccharides. Because of LacY, higher concentration of sugar molecules can be maintained inside a cell. Keeping higher level of sugar is essential because all bacteria must utilize the energy sources, such as carbohydrate, from their environment in order to produce ATP. ATP provides energy for the biosynthetic processes that bacteria use for their maintenance and reproduction. These processes are found in many organisms and play a crucial role in many aspects of cell function. Therefore, many biochemical techniques to study LacY have been applied to the study of many other similar membrane proteins. Since LacY has been studied as a model protein for many other secondary transporter proteins, it is important to understand the structural basis for this transporter because it may give us detailed understanding of energy transduction mechanisms utilized in many living organisms.

Structure
LacY has 12 transmembrane helices in which the N- and C-terminal 6 helices form two bundles. The 6 helices domains have a symmetrical arrangement to form a similar mirror image of each other and they are positioned to form a large interior hydrophilic cavity open to face the inside of a cell. All the available structures up to date exhibits an inward facing conformation and many studies have indicated that the periplasmic barrier is tightly closed in the absence of sugar binding. Therefore, the inward-facing conformation, which faces the cytoplasm, represents LacY's natural state in the membrane. A single β-D-galactopyranosyl1-thio-β-D-galactopyranoside (TDG) was bound to the cavity in the X-ray structure to mimic the actual sugar binding and there is only 1 binding site.

Mechanism
First, H+ binds to LacY, followed by lactose binding. A conformational change in LacY results in translocation of substrates and as they are released, LacY returns to its original conformation.

Sugar binding
Intensive mutagenic analysis of LacY has revealed that Glu126 and Arg144 are crucial for substrate binding and Glu269 is known to be involved in both H+ translocation and substrate binding. It has been postulated that Glu126 and Arg144 may interact with oxygen atoms of sugar via water molecules. Arg144 forms H-bonds with oxygen atoms of the galactopyranosyl ring. Glu126 may also interact with the oxygen atoms of the galactopyranosyl ring via water molecules. Furthermore, Trp151 may have a hydrophobic interaction with the galactopyranosyl ring. The hydrophobic interaction may orient the galactopyranosyl ring so that hydrogen bonds can be optimized. Glu269 and Arg144 form a salt bridge that can maintain the H-bond between Glu269 and Trp151 to keep Trp151 in the target orientation. Since Glu269 is also involved in H+ translocation, interaction between Glu269 in the C-terminal domain and Arg144 and Trp151 in the N- terminal domain may play a crucial role in the overall energy transduction mechanism that can change the overall conformation of LacY.

H+ translocation
His322, Glu325 and Arg302 are directly involved H+ translocation and it involves a series of protonation and deprotonation events. When LacY is in the outward facing conformation that faces the periplasm, H+ is on Glu269 or shared between Glu269 and His322. The sugar is recognized by Trp151, Arg144 and Glu126, which triggers H+ transfer to His322 and then to Glu325 as Glu269 is recruited to complete the binding site. As a result, transition to the inward-facing conformation is induced and sugar is then released into the cytoplasm, followed by release of H+. The release if H+ is induced by the change in pKa of Glu325 due to exposure to solvent in the hydrophilic cavity. After releasing the H+, transition back to the outward-facing conformation may be favored to accept another proton from periplasm.

How does proton gradient drive the reaction? H+ transfer network and the sugar-binding site is over 6 Å apart, hence they do not interact directly. In fact, proton gradient has negligible effect on binding affinity for lactose. In the presence of proton gradient, the rate-limiting step of the whole process is the dissociation of sugar or return of conformation back to the outward-facing. Therefore, proton gradient may improve the rate of deprotonation on the cytoplasmic side, and hence allows unloaded LacY to face the periplasmic side again. Thus, proton gradient directly affects the kinetics of the reaction rather than the actual physical binding of the sugar.

Summary
Countless attempts to engineer LacY in order to study its mechanism of energy transduction have expanded our knowledge about membrane proteins. It also gave us some unexpected answers. Since more types of membrane proteins are now purified and crystallized to give x-ray structures, our knowledge from LacY is likely to play an important role as we study the new proteins. To summarize, LacY couples the energy released from downhill transport of H+ to drive accumulation of lactose against a concentration gradient. Only an inward-facing conformation has been solved to this date and it shows that N- and C-terminal domains are positioned in a way to create a hydrophilic cavity in the middle. Many mutagenic studies have revealed the critical residues that play key roles during energy transduction. The most convincing model of mechanism to this date is that LacY exhibits an alternating access to the binding site that drives the conformation change as a proton and a sugar binds in an orderly fashion.